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Using naturally colored cotton (NCC) can eliminate dyeing, printing and industrial processing, and reduce sewage discharge and energy consumption. Proanthocyanidins (PAs), the primary coloration components in brown fibers, are polyphenols formed by oligomers or polymers of flavan-3-ol units derived from anthocyanidins. Three essential structural genes for flavanone and flavonoid hydroxylation encoding flavanone-3-hydroxylase (F3H), flavonoid 30 -hydroxylase (F30 H) and flavonoid 30 50 -hydroxylase (F30 50 H) are initially committed in the flavonoid biosynthesis pathway to produce common precursors. The three genes were all expressed predominantly in developing fibers of NCCs, and their expression patterns varied temporally and spatially among NCC varieties. In GhF3Hi, GhF30 Hi and GhF30 50 Hi silenced lines of NCC varieties XC20 and ZX1, the expression level of the three genes decreased in developing cotton fiber, negatively correlated with anthocyanidin content and fiber color depth. Fiber color depth and type in RNAi lines changed with endogenous gene silencing efficiency and expression pattern,the three hydroxylase genes functioned in fiber color formation. GhF3H showed functional differentiation among NCC varieties and GhF30 H acted in the accumulation of anthocyanin in fiber. Compared with GhF30 H, GhF30 50 H was expressed more highly in brown fiber with a longer duration of expression and caused lighter color of fibers in GhF30 50 H silenced lines. These three genes regulating fiber color depth and type could be used to improve these traits by genetic manipulation. [ABSTRACT FROM AUTHOR]